Optimized dengue virus entry inhibitory peptide (10an)

ABSTRACT

The invention relates peptide entry inhibitors and methods of determining such inhibitors that are bindable to regions of viruses having class II E proteins, such as the dengue virus E protein, as candidates for in vivo anti-viral compounds.

CROSS-REFERENCE TO RELATED APPLICATION

This is a national stage application of PCT/US2008/69725, filed Jul. 11,2008, to which this application claims priority from and any otherbenefit of U.S. provisional patent application Ser. No. 60/949,733 filedon Jul. 13, 2007, the entire disclosure of which is hereby incorporatedby reference.

GOVERNMENT SUPPORT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant no.GM068152 awarded by the National Institute of Health (NIH).

TECHNICAL FIELD

The invention relates to inhibitors that are bindable to regions of avirus. More particularly, the invention relates to inhibitors that arebindable to regions in the dengue virus envelope glycoprotein, or denguevirus E protein, which is a class II viral E protein. Even moreparticularly, the invention relates to peptide entry inhibitors andmethods of determining such inhibitors that are bindable to regions ofthe dengue virus E protein, as candidates for in vivo anti-viralcompounds.

BACKGROUND OF THE INVENTION

Dengue virus, a member of the flavivirus family, imposes one of thelargest social and economic burdens of any mosquito-borne viralpathogen. There is no specific treatment for infection, and control ofdengue virus by vaccination has proved elusive. Several otherflaviviruses are important human pathogens, including yellow fever, WestNile, tick-borne encephalitis (TBE) and Japanese encephalitis viruses(JE).

Enveloped viruses enter cells by membrane fusion. Structural protein Eof the flavivirus, which mediates both receptor binding and fusion, is aso-called “class II” viral fusion protein. Two classes of viral “fusionmachines” have been identified so far. Class I viral fusion proteinsinclude those of the myxo- and paramyxoviruses (e.g., influenza), theretroviruses (e.g., HIV), and the filoviruses (e.g., Ebola). Class IIfusion proteins are found in not only the flaviviruses (yellow fever,West Nile, etc.), but also the alphaviruses which includes SemlikiForest virus and Sindbis virus, as well as Hepatitis C. The structuralcharacteristics of the two classes are quite different, but bothaccomplish the same task, i.e. fusion of two lipid bilayers.

The more familiar class I fusion proteins, exemplified by thehaemagglutinin (HA) of influenza virus and gp120/gp41 of HIV, have a“fusion peptide” at or near the N-terminus of an internal cleavagepoint. This hydrophobic and glycine-rich segment, buried in thecleaved-primed trimer of the class I fusion protein, emerges when alarge-scale conformational rearrangement is triggered by low pH (in thecase of HA), receptor binding (in the case of gp120/gp41), or othercell-entry related signal. The likely sequence of events that followinclude an interaction of the fusion peptide with the target-cellmembrane and a refolding of the trimer. The latter step brings togetherthe fusion peptide and viral-membrane anchor, thereby drawing togetherthe cellular and viral membranes and initiating the bilayer fusionprocess.

The class II proteins, found so far in flaviviruses and alphaviruses,have evolved structurally different but mechanistically related fusionarchitecture. As in class I proteins, a proteolytic cleavage (of PrM toM in flaviviruses, or of pE2 to E2 in alphaviruses) yields maturevirions, with the fusion proteins in a metastable conformation, primedfor fusion. The fusion peptide, an internal loop at the tip of anelongated subdomain of the protein, is buried at a protein interface andbecomes exposed in the conformational change initiated by exposure tolow pH.

The mechanism of fusion of class II viral fusion proteins is notwell-understood, and there are no therapeutics that can specificallyinhibit the fusion of such proteins. Only the pre-fusion structures ofone flaviviral and one alphaviral envelope protein have been determinedto date. There is a need for entry inhibitors that can specificallyinhibit viral infection by flaviviruses, alphaviruses, and hepatitisviruses. Further, because fusion is a key step in viral infectivity, abetter understanding of the mechanism of class II envelope proteins,including the dengue virus envelope protein, and identification ofdruggable regions within such proteins will further development oftherapeutics that can specifically inhibit viral infection byflaviviruses, alphaviruses, and hepatitis viruses.

SUMMARY OF THE INVENTION

The invention provides peptide entry inhibitors that are bindable toregions in viral class II E proteins. The interaction of an inhibitorwith such regions, or the modulation of the activity of such regionswith an inhibitor, could inhibit viral fusion and hence viralinfectivity. In one aspect, the invention provides compounds and methodsof screening the compounds against these bindable regions in order todiscover therapeutic candidates for a disease caused by a virus having aclass II protein. Diseases for which a therapeutic candidate may bescreened include dengue fever, dengue hemorrhagic fever, tick-borneencephalitis, West Nile virus disease, yellow fever and hepatitis C.

In one embodiment, a method for identifying a therapeutic candidate fora disease caused by a virus having class II E protein, includescontacting a class II E protein which includes a bindable region with acompound, wherein binding of said compound indicates a therapeuticcandidate. Compounds may be selected from compounds including peptides.Binding may be assayed either in vitro or in vivo. In certainembodiments, the protein is dengue virus E protein. Such bindableregions also may be utilized in the structure determination, drugscreening, drug design, and other methods described and claimed herein.

Furthermore, the invention provides for methods of inhibiting viralinfection by dengue virus and/or binding between the virion envelope ofdengue viruses and membranes of the target cell (the process thatdelivers the viral genome into the cell cytoplasm). The inventionprovides for methods that employ peptides or peptide derivatives toinhibit dengue virus:cell binding. The invention provides for methods oftreatment of diseases induced by the dengue virus.

In another embodiment, a peptide entry inhibitor includes an amino acidsequence presented as SEQ ID NO: 1.

In yet another embodiment, a method for determining an inhibitor for avirus includes the steps of contacting a compound within a bindableregion of the virus, and determining the bindability of the compound tothe bindable region of virus, wherein the bindability of compoundmeasures inhibitory activity of the compound against the virus.

In still yet another embodiment, a method of treatment of dengue virusinfection includes the steps of administering a therapeuticallyeffective amount of a peptide, wherein the peptide inhibits dengue virusvirion:cell fusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the genomic organization of the dengue virus;

FIG. 2 is a graph representing the dose response inhibition curveagainst DENV-2 for the 10AN peptide;

FIG. 3 is a graph representing the dose response inhibition curveagainst DENV-2 for a scrambled sequence for the 10AN peptide; and

FIG. 4 is a graph representing the cytotoxicity assay of the 10ANpeptide at various concentrations.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention relates to methods of inhibiting dengueinfection that includes inhibiting the fusion between the virionenvelope and a cell membrane, the process that delivers the viral genomeinto the cell cytoplasm.

Any peptide or protein which inhibits the fusion between the denguevirion envelope and a cell membrane, including those of the dengue viruswhich infect human as well as nonhuman hosts, may be used according tothe invention. In various embodiments of the invention, these denguepeptide entry inhibitors may include, but are not limited to peptidesrelated to several membrane-interactive bindable regions of dengue virusproteins. The genomic organization of the dengue virus is illustrated inFIG. 1.

The term “bindable region”, when used in reference to a peptide, nucleicacid, complex and the like, refers to a region of a dengue virus Eprotein or other class II E protein which is a target or is a likelytarget for binding an agent that reduces or inhibits viral infectivity.For a peptide, a bindable region generally refers to a region whereinseveral amino acids of a peptide would be capable of interacting with atleast a portion of the dengue virus E protein. For a peptide or complexthereof, bindable regions including binding pockets and sites,interfaces between domains of a peptide or complex, surface grooves orcontours or surfaces of a peptide or complex which are capable ofparticipating in interactions with another molecule, such as a cellmembrane.

In one embodiment, the dengue peptide entry inhibitor is 10AN having aSEQ ID No. 1: FWFTLIKTQAKQPARYRRFC.

In another embodiment of the invention, peptides related to the denguepeptide entry inhibitor include homologous peptides. As used herein, theterm homologous dengue peptide entry inhibitors is to be interpreted aspeptides having a sequence identical to the corresponding portion of thedengue virus inhibitory protein and peptides in which one or more aminoacids are substituted by functionally equivalent amino acids. The termalso refers to derivatives of these peptides, including but not limitedto benzylated derivatives, glycosylated derivatives, and peptides whichinclude enantiomers of naturally occurring amino acids.

In other embodiments of the invention, the dengue peptide entryinhibitors, related peptides or derivatives are linked to a carriermolecule such as a protein. Proteins contemplated as being usefulaccording to this embodiment of the invention, include but are notlimited to, human serum albumen. Dengue peptide entry inhibitorscomprising additional amino acids are also contemplated as usefulaccording to the invention.

The dengue entry inhibitory peptides of the invention may be utilized toinhibit dengue virus virion:cell fusion and may, accordingly, be used inthe treatment of dengue virus infection in mammals. These mammals arepatients that may include, but are not limited to, humans, dogs, cats,birds, horses, etc. The peptides of the invention may be administered topatients in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. Methods for administering peptides to patients are well known tothose of skill in the art; they include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,oral, and intranasal. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intravenous injection. Otherembodiments contemplate the administration of the dengue entryinhibitory peptides or derivatives thereof, linked to a molecularcarrier including human serum albumin (HSA).

A number of techniques can be used to screen, identify, select anddesign chemical entities capable of associating with a dengue virus Eprotein or other class II E protein, structurally homologous molecules,and other molecules. Knowledge of the structure for a dengue virus Eprotein or other class II E protein, determined in accordance with themethods described herein, permits the design and/or identification ofmolecules and/or other modulators which have a shape complementary tothe conformation of a dengue virus E protein or other class II Eprotein, or more particularly, a druggable region thereof. It isunderstood that such techniques and methods may use, in addition to theexact structural coordinates and other information for a dengue virus Eprotein or other class II E protein, and structural equivalents thereof.

In one aspect, the method of drug design generally includescomputationally evaluating the potential of a selected chemical compoundto associate with a molecule or complex, for example any class II viralE protein. For example, this method may include the steps of employingcomputational means to perform a fitting operation between the selectedchemical compound and a bindable region of the molecule or complex andanalyzing the results of the fitting operation to quantify theassociation between the chemical entity and the bindable region.

In another aspect, candidates as dengue peptide entry inhibitors of DENVinfectivity that target the viral E protein were determined through theuse of primary amino acid sequence data in conjunction a Monte Carlobinding algorithm and a Wimley-White interfacial hydrophobicity scale.

The term “Monte Carlo,” as used herein, generally refers to anyreasonably random or quasi-random procedure for generating values ofallowed variables. Examples of Monte Carlo methods include choosingvalues: (a) randomly from allowed values; (b) via a quasi-randomsequence like LDS (Low Discrepancy Sequence); (c) randomly, but biasedwith experimental or theoretical a priori information; and (d) from anon-trivial distribution via a Markov sequence.

More particularly, a “Monte Carlo” method is a technique which obtains aprobabilistic approximation to the solution of a problem by usingstatistical sampling techniques. One Monte Carlo method is a Markovprocess, i.e., a series of random events in which the probability of anoccurrence of each event depends only on the immediately precedingoutcome. (See Kalos, M. H. and Whitlock, P. A. “Monte Carlo Methods:Volume I: Basics,” John Wiley & Sons, New York, 1986; and Frenkel, D.,and Smit, B. “Understanding Molecular Simulation: From Algorithms toApplications: Academic Press, San Diego, 1996).

The Wimley-White interfacial hydrophobicity scale is a tool forexploring the topology and other features of membrane proteins by meansof hydropathy plots based upon thermodynamic principles.

Materials and Methods Preparation of Dengue Peptide Entry Inhibitors

Peptides may be produced from naturally occurring or recombinant viralproteins, or may be produced using standard recombinant DNA techniques(e.g. the expression of peptide by a microorganism which containsrecombinant nucleic acid molecule encoding the desired peptide, underthe control of a suitable transcriptional promoter, and the harvestingof desired peptide from said microorganism). Preferably, the peptides ofthe invention may be synthesized using any methodology known in the art,including but not limited to, Merrifield solid phase synthesis(Clark-Lewis et al., 1986, Science 231:134-139).

Viruses and Cells

DENV-1 strain HI-1, DENV-2 strain NG-2, DENV-3 strain H-78, and DENV-4strain H-42 were obtained from R. Tesh at the World Health OrganizationArbovirus Reference Laboratory at the University of Texas at Galveston.Viruses were propagated in the African green monkey kidney epithelialcell line, LLCMK-2, a gift of K. Olsen at Colorado State University.LLCMK-2 cells were grown in Dulbecco's modified eagle medium (DMEM) with10% (v/v) fetal bovine serum (FBS), 2 mM Glutamax, 100 U/ml penicillinG, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B, at 37° C. with5% (v/v) CO₂.

Focus Forming Unit (FFU) Reduction Assay

LLCMK-2 target cells were seeded at a density of 1×10⁵ cells in eachwell of a E-well plate 24 h prior to infection. Approximately 200 FFU ofvirus were incubated with or without chemistries in serum-free DMEM for1 h at rt. Virus/chemistry or virus/control mixtures were allowed toinfect confluent target cell monolayers for 1 h at 37° C., with rockingevery 15 m, after which time the medium was aspirated and overlaid withfresh DMEM/10% (v/v) FBS containing 0.85% (w/v) Sea-Plaque Agarose(Cambrex Bio Science, Rockland, Me.). Cells with agar overlays wereincubated at 4° C. for 20 m to set the agar. Infected cells were thenincubated at 37° C. with 5% CO₂ for 3 days (DENV-1, 3 and 4) or 5 days(DENV-2). Infected cultures were fixed with 10% formalin overnight at 4°C., permeabilized with 70% (v/v) ethanol for 20 m, and rinsed with PBSprior to immunostaining. Virus foci were detected using supernatant frommouse anti-DENV hybridoma E60 (obtained from M. Diamond at WashingtonUniversity) followed by horseradish peroxidase-conjugated goatanti-mouse immunoglobulin (Pierce, Rockford, Ill.) and developed usingAEC chromogen substrate (Dako, Carpinteria, Calif.). Results wereexpressed as the average of at least two independent trials with threereplicates in each trial.

Cytoxicity Assay

The cytotoxicity of the chemistries was measured by monitoringmitochondrial reductase activity using the TACS™ MTT cell proliferationassay (R&D Systems, Inc., Minneapolis, Minn.) according to themanufacturer's instructions. Dilutions of chemistries in serum-free DMEMwere added to confluent monolayers of LLCMK-2 cells in 96-well platesfor 1 h at 37° C., similar to the focus forming inhibition assays, andsubsequently incubated at 37° C. with 5% (v/v) CO₂ for 24 h. Absorbanceat 560 nm was measured using a Tecan GeniosPro plate reader (Tecan US,Durham, N.C.).

Mechanistic Assays with 10AN

Post-Entry Focus-Forming Assay with 10AN Against DENV-2

To determine if the observed inhibitory effect was due to interferencewith post-entry steps in the viral life cycle, approximately 200 FFU ofDENV-2 without 10AN was allowed to bind and enter target cells for 1 hat 37° C. as described for the focus forming assay. Unbound virus wasthen removed by rinsing with PBS and 10AN was added to the cellspost-entry for 1 hr at 37° C. Cultures were washed again in PBS andagarose overlays, incubation, and immunological detection was conductedas described for the focus forming assay.

Pre-Binding Focus-Forming Assay with 10AN Against DENV-2

To determine if the observed inhibitory effect was due to interferencecaused by modifications to the target cell surface, 10AN was incubatedwith the target cells for 1 h at 4° C., the cells were rinsed with PBS,and approximately 200 FFU of DENV-2 was allowed to infect the cells at4° C. Agarose overlays, incubation, and immunological detection wereconducted as described for the focus forming assay.

Post-Binding Focus-Forming Assay with 10AN Against DENV-2

To determine if the observed inhibitory effect was due to interferencewith interactions that occur pre-binding versus post-binding of virionsto the target cells, approximately 200 FFU of DENV-2 was allowed to bindto target LLCMK-2 cells for 1 h at 4° C. to allow binding, but preventinternalization. Unbound virus was washed off with PBS at 4° C., then CF238 was added and incubated at 4° C. for 1 h. Cultures were washed againin 4° C. PBS and warmed to 37° C. Agarose overlays, incubation, andimmunological detection were conducted as described for the focusforming assay.

qRT-PCR Virus Binding Assay

Infection of LLCMK-2 target cells in six well plates was performed induplicate using 10⁵ FFU of DENV-2 that had been pre-incubated for 45 mat 4° C. with CF 238 or pooled heterotypic anti-DENV human serum. Aftera 45 m infection at 4° C., infected monolayers were washed with PBS andharvested with a cell scraper, added to a 1.5 ml microfuge tubecontaining 350 μl of AR-200 silicone oil (Sigma-Aldrich, St. Louis, Mo.)mixed with 150 μl of silicone fluid (Thomas, Swedesboro, N.J.), and spunat 14,000 rpm in a microfuge for 1 m to separate the unbound virus fromthe cell-bound virus in the pellets. The tubes were then submerged inliquid nitrogen for 30 s to freeze the contents. The cell pellets withbound virus were recovered by clipping off the bottoms of the tubes withsmall wire clippers into 15 ml conical tubes. Viral RNA was extractedfrom the cell pellets using the Qiagen Viral RNA Extraction kit (Qiagen,Chatsworth, Calif.).

Quantitative real time reverse transcription PCR (qRT-PCR) was performedon the extracted RNA using the Quantitect Sybr Green RT-PCR kit (Qiageninc., Chatsworth, Calif.), following the manufacturer's specificationsand amplification protocols, using dengue-specific primers: (Den2F:catatgggtggaatctagtacg, Den2R: catatgggtggaatctagtacg). Each reactionwas performed in 20 μL total volume (10 μL 2×SYBR green master mix, 0.5μL of 10 μM of each primer, 0.2 μL reverse transcriptase, and 5 μL viralRNA) using a Lightcycler thermal cycler (Roche Diagnostics, Carlsbad,Calif.), and according to the following amplification protocol: 50° C.for 20 min to reverse transcribe the RNA; 95° C. for 15 min to activatethe HotStart Taq DNA Polymerase; 45 PCR cycles: 94° C. for 15s, 50° C.for 15s, 72° C. for 30s, the last step was also the fluorescence dataacquisition step. Melting curve analysis was performed by a slowincrease in temperature (0.1° C./s) up to 95° C. The threshold cycle,representing the number of cycles at which the fluorescence of theamplified product was significantly above background, was calculatedusing Lightcycler 5.3.2 software (Roche).

Analysis

Figures were generated using the Origin 6.0 graphing software(Northampton, Mass.). Statistical analyses were performed using theGraphpad Prism 4.0 software package (San Diego, Calif.). P values lessthan 0.05 were considered significant.

Results

Inhibition Assays with Different Chemistries Against DENV-2

Focus-forming assays were used to quantitate the inhibitory activitiesof each chemistry against DENV-2 as previously described (Hrobowski, etal, 2005). As seen in FIG. 2, dose response curves were generated overconcentration ranges dictated by the solubilities of the chemistries in1% DMSO/aqueous solution. Control 1% DMSO/PBS solutions showed no DENVinhibitory activity in this assay system (data not shown). The 10ANpeptide showed an increase in inhibitory activity as a function ofconcentration. As seen in FIG. 3, a scrambled sequence of the 10ANpeptide showed no consistent activity directed towards inhibitionagainst DENV-2.

Cytotoxicity

To determine if the observed DENV inhibition effects were due tocellular toxicity that impacted viral replication, the effect of thechemistry on the mitochondrial reductase activity of the target cellswas measured over the concentration ranges that showed viral inhibition.In confluent cell monolayers that replicated the conditions in the focusforming assays no sign of toxicity was observed with any compoundcompared to medium only controls (p>0.05, ANOVA with Dunnett's posthoctest) as seen in FIG. 4. Thus, the inhibitory activity of the 10ANpeptide is not due to toxicity.

Pre-Binding Focus-Forming Assay with 10AN Against DENV-2

In this assay, 10AN was added to target cells for 1 h prior to infectionwith DENV-2 to determine if 10AN inhibits entry through interactiondirectly with the target cells. Treatment of target cells with 10ANprior to DENV-2 infection resulted in no evidence of inhibition,indicating that 10AN does not function by interacting with or modifyingthe target cell surface, and must be present along with the virus inorder to inhibit entry.

qRT-PCR Virus Binding Assay with 10AN Against DENV-2

In order to directly test if 10AN interferes with virus binding totarget cells, binding assays were conducted using qRT-PCR to monitorattachment of virus to target cells. In these experiments, virus wasco-incubated with 10AN for 45 m at 4° C. and used to infect target cellsat 4° C. for 45 m. The cells were then scraped off the plates andcentrifuged through an oil mixture with a density that allowed passageof the cells, but not free virus, to the bottom of the tube. RNA wasthen extracted from the cell pellets and amplified with DENV-2 specificprimers. Pre-incubation of DENV-2 with 10AN did not inhibit virusbinding, as measured by the qRT-PCR signal, whereas pre-incubation ofDENV-2 with pooled human heterotypic anti-DENV-2 serum resulted in alarge decrease in the attachment of virus to target cells. Thisindicates that 10AN does not prevent virus binding/attachment to targetcells under the experimental condition tested.

Based upon the foregoing disclosure, it should now be apparent that theuse of peptide entry inhibitors that are bindable to regions of thedengue virus E protein, as potential candidates for the development ofanti-viral compounds as described herein will carry out the objects setforth hereinabove. It is, therefore, to be understood that anyvariations evident fall within the scope of the claimed invention andthus, the selection of specific component elements can be determinedwithout departing from the spirit of the invention herein disclosed anddescribed.

1. A peptide entry inhibitor comprising: an amino acid sequencepresented as SEQ ID NO:
 1. 2. The peptide entry inhibitor of claim 1,wherein the peptide entry inhibitor inhibits a class II envelopeprotein.
 3. The peptide entry inhibitor of claim 2, wherein the class IIenvelope protein is a dengue virus E protein.
 4. The peptide entryinhibitor of claim 1, wherein the peptide entry inhibitor inhibitsdengue virus virion:cell fusion.
 5. A pharmaceutical compositioncomprising the peptide entry inhibitor of claim
 1. 6. The pharmaceuticalcomposition of claim 5 further comprising a biocompatible carrier. 7.The pharmaceutical composition of claim 6, wherein the biocompatiblecarrier is selected from the group consisting of saline, bufferedsaline, dextrose and water.
 8. A method for determining an inhibitor fora virus, the method comprising the steps of: contacting a compoundwithin a bindable region of the virus; and determining the bindabilityof the compound to the bindable region of virus, wherein the bindabilityof compound measures inhibitory activity of the compound against thevirus.
 9. The method of claim 8, wherein the compound is a peptide. 10.The method of claim 9, wherein the peptide is a linear peptide.
 11. Themethod of claim 10, wherein the peptide is a peptide entry inhibitor fora class II envelope protein.
 12. The peptide of claim 11, wherein theclass II envelope protein is a dengue virus E protein.
 13. The method ofclaim 9, wherein the peptide has an amino acid sequence presented as SEQID NO:
 1. 14. A method of treatment of dengue virus infection comprisingthe steps of: administering a therapeutically effective amount of apeptide, wherein the peptide inhibits dengue virus virion:cell fusion.15. The method of claim 14, wherein the peptide has an amino acidsequence presented as SEQ ID NO:
 1. 16. The method of claim 14, whereinthe peptide is administered selected from the group consisting ofintradermally, intramuscularly, intraperitoneally, intravenously,subcutaneously, orally, and intranasally.
 17. The method of claim 14,wherein the peptide is linked to a molecular carrier.
 18. The method ofclaim 17, wherein the molecular carrier is human serum albumin (HSA).19. The method of claim 14, wherein the peptide is administered to amammal.